The invention relates to new serine protease inhibitors, pharmaceutical compositions containing the same, as well as to the use of said inhibitors for the manufacture of a medicament for treating and preventing thrombin-related diseases.
Serine proteases are enzymes which, amongst other things, play an important role in the blood coagulation cascade. Members of this group of proteases are for example thrombin, trypsin, factors VIIa, IXa, Xa, XIa, XIIa, and protein C.
Thrombin is the serine protease which regulates the last step in the coagulation cascade. The prime function of thrombin is the cleavage of fibrinogen to generate fibrin monomers, which form an insoluble gel by cross-linking. In addition, thrombin regulates its own production by activating factors V and VIII earlier in the cascade. It also has important actions at the cellular level, where it acts on specific receptors to cause platelet aggregation, endothelial cell activation and fibroblast proliferation. Thus thrombin has a central regulatory role in haemostasis and thrombus formation. Since inhibitors of thrombin may have a wide range of therapeutical applications, extensive research has been performed in this area. In the development of synthetic inhibitors of serine proteases, and more specifically of thrombin, the interest in small synthetic peptides that are recognized by proteolytic enzymes in a manner similar to that of natural substrates, has increased. As a result, new peptide-like inhibitors have been prepared, such as the transition state inhibitors of thrombin.
The search for more effective and more selective thrombin inhibitors continues unabated in order to obtain thrombin inhibitors which can be administered in lower dosages and which have fewer and less severe side effects. Furthermore, special attention is paid to oral bioavailability. Potent intravenous thrombin inhibitors are clinically effective in acute care settings requiring the treatment of thrombin-related diseases. However, particularly the prevention of thrombin-related diseases such as myocardial infarct, thrombosis and stroke require long-term therapy, preferably by orally dosing an anticoagulant.
Many of the peptide-like serine protease inhibitors, in particular thrombin inhibitors, disclosed in prior publications are based on the sequence -D-Phe-Pro-Arg-, see for example compounds as disclosed by Brady et al. [Bioorganic and Medical Chemistry, 3 (1995), 1063-78] and in U.S. Pat. No. 5,597,804. Thrombin inhibitors may also contain lysine side chains instead of arginine, such as other inhibitors disclosed by Brady et al., and Lewis et al. [Thrombosis and Haemostasis, 74(4) (1995), 1107-12], and further by Jones et al. [J. Enzyme Inhibition, 9 (995), 43-60]. In the latter publication it was reported that tripeptide compounds containing xcex1-keto methyl ester functions are labile compounds and therefore unfavourable for further development as thrombin inhibitors. Also thrombin inhibitors having an aminocyclohexyl moiety instead of lysine or arginine side chain are known [WO 94/25051]. From these and also other references [e.g. U.S. Pat. No. 5,523,308] a number of variations at the C-terminus of these peptide-like thrombin inhibitors is known. The developments in this field have already improved the understanding of how to modulate the biological properties of this type of thrombin inhibitors. However, although great effort is being spend on finding selective thrombin inhibitors having good oral bioavailability there are still few transition state thrombin inhibitors known in the art which fulfill these requirements.
Surprisingly, it has now been found that compounds of the formula:
R1SO2xe2x80x94Bxe2x80x94Xxe2x80x94Zxe2x80x94C(O)xe2x80x94Yxe2x80x83xe2x80x83(I)
wherein
R1 is R2OOCxe2x80x94(CHR2)mxe2x80x94 or R2NHxe2x80x94COxe2x80x94(CHR2)mxe2x80x94 or is selected from (1-12C)alkyl, (2-12C)alkenyl, which groups may optionally be substituted with (3-12C)cycloalkyl, (1-6C)alkoxy, OH, COOR2, CF3 or halogen, and from (6-14C)aryl, (7-15C)aralkyl and (8-16)aralkenyl, the aryl groups of which may optionally be substituted with (1-6C)alkyl, (3-8C)cycloalkyl, (1-6C)alkoxy, OH, COOH, CF3 or halogen;
m is 1, 2 or 3;
each group R2 is independently H, (1-12C)alkyl, (3-8C)cycloalkyl, (6-14C)aryl or (7-1 5C)aralkyl, the aryl groups of which may be substituted with (1-6C)alkyl, (1-6C)alkoxy or halogen;
B is a bond, an amino-acid of the formula xe2x80x94NHxe2x80x94CH[(CH2)pC(O)OH]xe2x80x94C(O)xe2x80x94 or an ester derivative thereof wherein p is 1, 2 or 3, Gly, D-1-Piq, D-3-Piq, D-1-Tiq, D-3-Tiq, D-Atc, Aic, or a L- or D-amino acid having a hydrophobic, basic or neutral side chain;
X is an amino acid with a hydrophobic side chain, glutamine, serine, threonine, a cyclic amino acid optionally containing an additional heteroatom selected from N, O or S, and optionally substituted with (1-6C)alkyl, (1-6C)alkoxy, benzyloxy or oxo, or X is 2-amino-isobutyric acid, xe2x80x94NR2xe2x80x94CH2xe2x80x94C(O)xe2x80x94 or the fragment 
xe2x80x83wherein n is 2, 3, or 4, W is CH or N and R3 is H, (1-6C)alkyl or phenyl which groups may optionally be substituted with hydroxy, (1-6C)alkoxy, COOH, COO(1-6C)alkyl, CONH2, or halogen;
Z is lysine or 4-aminocyclohexylglycine;
Y is xe2x80x94NH-(1-6C)alkylene-C6H5, the phenyl group of which may be substituted with (1-6C)alkyl, (1-6C)alkoxy or halogen, or Y is xe2x80x94OR4 or xe2x80x94NR5R6, wherein R4 is H, (2-6C)alkyl or benzyl, and R5 and R6 are independently H, (1-6C)alkoxy, or (1-6C)alkyl optionally substituted with halogen, or R5 and R6 together are (3-6C)alkylene, or R5 and R6 together with the nitrogen atom to which they are bonded are 
xe2x80x83wherein V is O, S or SO2;
or a prodrug thereof or a pharmaceutically acceptable salt thereof, are potent and selective serine protease inhibitors. Specifically, the compounds of the present invention are inhibitors of thrombin, of factor VIIa/tissue factor and of factor Xa. Compounds of the invention show improved pharmacokinetics, and in particular good bioavailability after oral administration. The xcex1-(2-6C)keto ester compounds which are part of the present invention do not show the disadvantages of the previously reported labile xcex1-keto methyl ester compounds.
The compounds of the present invention are useful for treating and preventing thrombin-mediated and thrombin-associated diseases. This includes a number of thrombotic and prothrombotic states in which the coagulation cascade is activated which include, but are not limited to, deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterial occlusion from thrombosis or embolism, arterial reocclusion during or after angioplasty or thrombolysis, restenosis following arterial injury or invasive cardiological procedures, postoperative venous thrombosis or embolism, acute or chronic atherosclerosis, stroke, myocardial infarction, cancer and metastasis, and neurodegenerative diseases. The compounds of the invention may also be used as anticoagulants in extracorporeal blood circuits, as necessary in dialysis and surgery. The compounds of the invention may also be used as in vitro anticoagulants.
Preferred serine protease inhibitors according to this invention are the compounds wherein Z is lysine. More preferred are the compounds wherein X is a cyclic amino acid, an amino acid with a hydrophobic side chain, glutamine, serine, threonine, xe2x80x94NR2xe2x80x94CH2xe2x80x94C(O)xe2x80x94, or the fragment 
wherein R3 is H, (1-6C)alkyl or phenyl.
Particularly preferred are the compounds wherein X is proline, leucine, glutamine, threonine, phenylalanine, xe2x80x94NR2xe2x80x94CH2xe2x80x94C(O)xe2x80x94 wherein R2 is methyl, cyclopentyl or cyclohexyl, or the fragment 
wherein R3 is H or methyl.
Other preferred compounds are those wherein B is a bond or a D-amino acid having a hydrophobic or neutral side chain. The most preferred compounds of the invention are those wherein R1 is (1-6C)alkyl or benzyl. Preferably R4 in the definition of Y is (2-6C)alkyl or benzyl. In particular preferred are the compounds wherein Y is xe2x80x94OCH(CH3)2. Also preferred compounds have Y is NH2.
The term (1-12C)alkyl means a branched or unbranched alkyl group having 1 to 12 carbon atoms, such as methyl, ethyl, t-butyl, isopentyl, heptyl, dodecyl, and the like. Preferred alkyl groups are (1-6C)alkyl groups, having 1-6 carbon atoms.
A (2-12C)alkenyl group is a branched or unbranched unsaturated hydrocarbon group having 2 to 12 carbon atoms. Preferred are (2-6C)alkenyl groups. Examples are ethenyl, propenyl, allyl, and the like.
The term (1-6C)alkylene means a branched or unbranched alkylene group having 1 to 6 carbon atoms, such as xe2x80x94(CH2)sxe2x80x94 and s is 1 to 6, xe2x80x94CH(CH3)xe2x80x94, xe2x80x94CH(CH3)xe2x80x94(CH2)xe2x80x94, etc. Preferred alkylene groups in the definition of Y are ethylene and methylene.
The term (1-6C)alkoxy means an alkoxy group having 1-6 carbon atoms, the alkyl moiety of which has the meaning as previously defined.
The term (3-12C)cycloalkyl means a mono- or bicycloalkyl group having 3-12 carbon atoms which cycloalkyl group may optionally be substituted with an oxo group. Preferred are (3-8C)cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclo-octyl, etc. Cyclopentyl and cyclohexyl are even more preferred cycloalkyl groups. A preferred cycloalkyl substituted alkyl group in the definition of R1 is the camphor group.
A (6-14C)aryl group is an aromatic moiety of 6 to 14 carbon atoms. The aryl group may further contain one or more hetero atoms, such as N, S, or O. Examples of aryl groups are phenyl, naphthyl, (iso)quinolyl, indanyl, and the like.
(7-1 5C)Aralkyl and (8-16C)aralkenyl groups are alkyl and alkenyl groups respectively, substituted by one or more aryl groups, the total number of carbon atoms being 7 to 15 and 8 to 16, respectively.
The term halogen means fluorine, chlorine, bromine or iodine.
The term ester derivative means any appropriate ester derivative, preferably (1-4C)alkyl-esters, such as methyl-, ethyl- or t-butyl-esters.
The terms Atc means 2-aminotetralin-2-carboxylic acid and Aic means amino indane carboxylic acid. The terms 1- and 3-Tiq mean 1,2,3,4-tetrahydroisoquinoline-1- and -3-carboxylic acid, respectively; 1- and 3-Piq are perhydroisoquinoline-1- and -3-carboxylic acid, respectively.
The term amino acid having a hydrophobic side chain means an amino acid having a side chain being (3-8C)cycloalkyl, (6-14C)aryl or (1-6C)alkyl, which alkyl group may optionally be substituted with one or more (3-8C)cycloalkyl groups or (6-14C)aryl groups. The hydrophobic side chain may optionally be substituted with one or more substituents, such as hydroxy, halogen, trifluoromethyl, xe2x80x94OSO2CF3, (1-4C)alkyl (for instance methyl or ethyl), (1-4C)alkoxy (for instance methoxy), phenyloxy, benzyloxy, and the like. Preferred amino acids with a hydrophobic side chain are leucine, valine, cyclohexylalanine, 4-methoxy-cyclohexylalanine, cyclo-octylalanine, phenylalanine, D-naphthylalanine, tyrosine, O-methyl tyrosine (or: p-methoxy-phenylalanine), 3,3-diphenylalanine, norleucine and leucine.
Amino acids having a basic side chain are for example, but not limited to, arginine and lysine, preferably arginine.
The term amino acids having a neutral side chain refers to amino acids such as glutamine (Gln), methionine sulfon, asparagine (Asn) and the like. Preferred are Gln and Asn.
Cyclic amino acids are for example 2-azetidine carboxylic acid, proline, pipecolic acid, 1-amino-1-carboxy-(3-8C)cycloalkane (preferably 4C, 5C or 6C), 4-piperidine carboxylic acid, 4-thiazolidine carboxylic acid, 3,4-dehydro-proline, azaproline, 2-octahydroindole carboxylic acid, and the like. Preferred are 2-azetidine carboxylic acid, proline, pipecolic acid, 4-thiazolidine carboxylic acid, 3,4-dehydro-proline and 2-octahydroindole carboxylic acid. In the definitions, the term substituted means: substituted by one or more substituents.
The invention also includes prodrugs of the compounds of formula I, which after administration are metabolized into the active compounds. Suitable prodrugs are for example N-alkoxycarbonyl protected (preferably N-ethoxycarbonyl) derivatives of the compounds of formula I.
The invention further includes a process for preparing a compound of formula I, comprising coupling of suitably protected amino acids or amino acid analogs, followed by removing the protective groups.
The compounds according to formula I may be prepared in a manner conventional for such compounds. To that end, suitably Nac protected (and side-chain protected if reactive side-chains are present) amino acid derivatives or peptides are activated and coupled to suitably carboxyl protected amino acid or peptide derivatives either in solution or on a solid support. Protection of the xcex1-amino functions generally takes place by urethane functions such as the acid-labile tert-butyloxycarbonyl group (Boc), benzyloxycarbonyl (Cbz) group and substituted analogs or the base-labile 9-fluorenyl-methyloxycarbonyl (Fmoc) group. The Cbz group can also be removed by catalytic hydrogenation. Other suitable amino protective groups include Nps, Bpoc, Msc, etc. A good overview of amino protective groups is given is given in The Peptides, Analysis, Synthesis, Biology, Vol. 3 E. Gross and J. Meienhofer, Eds., (Academic Press, New York, 1981). Protection of carboxyl groups can take place by ester formation e.g. base-labile esters like methyl- or ethylesters, acid labile esters like tert-butylesters, or hydrogenolytically-labile esters like benzylesters. Protection of the side chain function of lysine or 4-aminocyclohexylglycine may be accomplished by using the aforementioned groups. Activation of the carboxyl group of the suitably protected amino acids or peptides can take place by the azide, mixed anhydride, active ester, or carbodiimide method, especially with the addition of catalytic and racemization-suppressing compounds like 1-hydroxybenzotriazole, N-hydroxysuccinimide, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, N-hydroxy-5-nor-bornene-2,3-dicarboximide. See, e.g. The Peptides, Analysis, Synthesis, Biology (see above) and Pure and Applied Chem. 59(3), 331-344 (1987).
The compounds of the invention, which can be in the form of a free base, may be isolated from the reaction mixture in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salts may also be obtained by treating the free base of formula I with an organic or inorganic acid such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, maleic acid, malonic acid, methanesulfonic acid, fumaric acid, succinic acid, tartaric acid, citric acid, benzoic acid, and ascorbic acid.
The compounds of this invention possess one or more chiral carbon atoms, and may therefore be obtained as a pure enantiomer, or as a mixture of enantiomers, or as a mixture containing diastereomers. Methods for obtaining the pure enantiomers are well known in the art, e.g. crystallization of salts which are obtained from optically active acids and the racemic mixture, or chromatography using chiral columns. For diastereomers straight phase or reversed phase columns may be used.
The compounds of the invention may be administered enterally or parenterally, and for humans Preferably in a daily dosage of 0.001-100 mg per kg body weight, preferably 0.01-10 mg per kg body weight. Mixed with pharmaceutically suitable auxiliaries, e.g. as described in the standard reference, Gennaro et al., Remington""s Pharmaceutical Sciences, (18th ed., Mack Publishing company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture) the compounds may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds can also be applied in the form of a solution, suspension, emulsion, e.g. for use as an injection preparation, or as a spray, e.g. for use as a nasal spray.
For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. Suitable carriers with which the compositions can be administered include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.
The invention is further explained by reference to the following illustrative Examples.
General
Abbreviations
Et=ethyl
Bzl=benzyl
Boc=tert-butyloxycarbonyl
Cbz=benzyloxycarbonyl
Cha=cyclohexylalanyl
Pro=prolyl
Lys=lysyl
Acg=4-aminocyclohexyl glycyl
TFA=trifluoro acetic acid
Pac=phenylacetyl
Nps=nitrophenylsulfonyl
Bpoc=2-p-biphenylisopropyloxycarbonyl
Asp=aspartyl
Glu=glutamyl
Dpa=diphenylalanyl
H-Aad-OH=amino-adipic acid
Tyr(Me)=(O-methyl)-tyrosyl
Phe=phenylalanyl
Nal=naphthylen-2-yl-alaninyl
H-3-Tiq-OH=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
Msc=methylsulfonylethyloxycarbonyl
Teoc=2-(trimethylsilyl)ethoxycarbonyl
norLeu(cyclo)-Gly-OH=(S)-3-amino-2-oxo-hexahydro-1-azepineacetic acid
norVal(cyclo)-Gly-OH=(S)-3-amino-2-oxo-1-piperidineacetic acid
Experimental
The solvent systems used in HPLC are: A: 0.5 M phosphate buffer pH=2.1; B: water; C: acetonitrile/water 3/2 v/v.
Unless stated otherwise the retention times (Rt (LC)) were determined on an analytical HPLC Supelcosil LC-18-DB column (5 xcexcm particles; 250xc3x972.1 mm), which was eluted using a gradient (as specified) of solvent systems A, B and C at a flow rate of 0.25 ml/min at 35xc2x0 C.